Alternative splicing of CARM1 regulated by LincGET-guided paraspeckles biases the first cell fate in mammalian early embryos

The heterogeneity of CARM1 controls first cell fate bias during early mouse development. However, how this heterogeneity is established is unknown. Here, we show that Carm1 mRNA is of a variety of specific exon-skipping splicing (ESS) isoforms in mouse two-cell to four-cell embryos that contribute to CARM1 heterogeneity. Disruption of paraspeckles promotes the ESS of Carm1 precursor mRNAs (pre-mRNAs). LincGET, but not Neat1, is required for paraspeckle assembly and inhibits the ESS of Carm1 pre-mRNAs in mouse two-cell to four-cell embryos. We further find that LincGET recruits paraspeckles to the Carm1 gene locus through HNRNPU. Interestingly, PCBP1 binds the Carm1 pre-mRNAs and promotes its ESS in the absence of LincGET. Finally, we find that the ESS seen in mouse two-cell to four-cell embryos decreases CARM1 protein levels and leads to trophectoderm fate bias. Our findings demonstrate that alternative splicing of CARM1 has an important role in first cell fate determination.


Supplementary Discussion
Besides, we analyzed the SINE, LINE, and LTR elements in Carm1 gene locus among mice, rats, rabbits, dogs, pigs, monkeys, and humans, and found these elements are conserved in Carm1 gene locus among these mammalian species (Extended Data Fig. 15).It raised the possibility that the model in which CARM1 heterogeneity is partially controlled by retrotransposon-associated lincRNA is conserved in other mammals.
The localization of many subnuclear speckles is regulated by their lncRNA component 1 .
Generally, paraspeckles in somatic cells are built around the lncRNA Neat1.Neat1 is not the only lncRNA involved in paraspeckles, for example, another lncRNA termed CTN-RNA, localizes specifically to paraspeckles in a number of cell types 2 .Besides, in human pluripotent cells, NEAT1 is poorly expressed and paraspeckles are barely detected 3 .This indicated that besides Neat1, paraspeckle components might assemble around other lncRNAs in different cell types for different functions.Interestingly, Neat1 ablation in mouse two-to four-cell embryos resulted in re-localization of partial NONO from paraspeckles around the nucleoli 4 rather than disrupting paraspeckles completely, while Neat1 ablation in HeLa and NIH-3T3 cells led to disappearance of paraspeckle that all NONO diffused throughout the nucleoplasm 5 .Besides, paraspeckles in somatic cells form near Neat1 gene loci 6 , but we here and others 4 found that paraspeckle components in mouse two-to fourcell embryos distribute evenly throughout the nucleoplasm.Our data here indicate that LincGET is the major lincRNA component involved in the assembly of paraspeckles in mouse two-to four-cell embryos and is essential for the localization of paraspeckles to the Carm1 gene locus.LincGET depletion led to the disappearance of paraspeckles in mouse early embryos, which is similar to Neat1 ablation for paraspeckles in HeLa and NIH-3T3 cells 5 .The involvement of the early embryo-specific LincGET in the assembly of paraspeckles in mouse two-to four-cell embryos reveals the highly specialized function and regulation of paraspeckles in early embryos, highlighting the cell-type specific function of paraspeckles through different lncRNA.
Our results indicated that PCBP1 accumulates in the nucleus of mouse late two-and early fourcell embryos (Extended Data Fig. 9a, b), which may be responsible for driving the change in alternative splicing under physiological conditions during late two-to early four-cell stage under the major wave of zygotic genome activation (ZGA).LincGET, which is specifically expressed in the nucleus of mouse late two-and early four-cell embryos, may work as a balancing mechanism to prevent the alternative splicing from being excessive.
It is reported that a lot of splicing factors, such as SRAF3, undergo exon-skipping splicing in mouse early embryos, especially at the two-cell stage 7 .CARM1 is also reported as one client protein of paraspeckles that can influence the paraspeckle organization in two-to four-cell embryos 4 .Both elevating CARM1 levels and inhibiting CARM1 impair the organization of paraspeckles, which suggests that CARM1 might feedback and regulate its expression level via alternative splicing.It is consistent with the observation that there is a regulatory cascade of alternative splicing events where some key splicing regulators are controlled by alternative splicing of their gene transcripts 7 .This may be the reason why alternative splicing is extremely complicated and extraordinary during preimplantation development.This study reveals that the high exon-skipping splicing promoting activity can be partially counteracted by the LincGET-guided paraspeckles in a locus-specific manner, providing mechanistic insight into the negative regulation side of exon-skipping splicing in mouse early embryos.In addition, the mechanisms responsible for the strong promotion of exon-skipping splicing beyond Pcbp1/2 in mouse early embryos is a fascinating question to be explored in the future.
We found that Pcbp1/2 silencing doesn't significantly alter TE fate, which may be due to that depletion of Pcbp1/2 didn't affect CARM1 protein level (Fig. 5g), even though Pcbp1/2 depletion decreased the exon-skipping splicing of Carm1 pre-mRNAs in mouse four-cell embryos (Fig. 4c, Extended Data Fig. 9e).PCBP1, plays important roles in both transcription regulation and translation control 8 .PCBP1 binds the 3'-untranslational region (3'-UTR) of p27 Kip1 mRNA, increases its stability, and enhances its translation 9 .The evidence indicates that PCBP1 not only can downregulate protein level through promoting exon-skipping splicing of its binding mRNAs, but also can upregulate protein level through enhance translation of its binding mRNAs.
LincGET-guided paraspeckles to the Carm1 genome locus requires HNRNPU.The DNA binding and RNA binding ability of HNRNPU gives it an essential role in the high-order arrangement of the nucleus 10 .For example, HNRNPU is necessary for the correct localization of the X chromosome inactivation specific transcript Xist to establish gene silencing and form the highly structured chromatin territories [11][12][13] .Whether LincGET participates in high order nuclear organization is an interesting direction to explore in the future.
We found that the noS and E5S of Carm1 pre-mRNAs are translated into protein, while the E3S, E6S, E56S, and E3456S cannot be translated into protein (Extended Data Fig. 3e), suggesting that the exon-skipping splicing events do cause frame shift of the CARM1 protein during translation.It is known that the extra stop codon generated by frame shift will activate the non-sense mediated mRNA decay (NMD) pathway, a selective RNA turnover mechanism to control steady-state RNA levels 14 .
Although the exon-skipping splicing events cause the decrease of CARM1 protein level, we found the mRNA level of CARM1 remained stable at two-to four-cell stage (Fig. 1c, Extended Data Fig. 3c, d), thus whether and how the NMD pathway is involved in balancing between RNA synthesis and RNA turnover to control RNA levels during early embryonic development is a field worthy of extensive study in the future work.